The present invention is generally in the field of bioelectronics and concerns electrically conducting solid matrices (to be referred to herein as xe2x80x9celectrodesxe2x80x9d) carrying redox enzymes such that an electric charge can flow between the surface of the electrode and the enzymes rendering them catalytically active. Also provided by the invention is a process for the preparation of the electrodes as well as devices, systems and methods making use of such electrodes. In accordance with one embodiment, the invention is applied for the determination of the presence and optionally the concentration of an analyte in a liquid medium. In accordance with another embodiment, the immobilized enzymes can be switched by light into two distinct biocatalytic states thus allowing the transduction and amplification of recorded optical signals thus fulfilling xe2x80x9creadxe2x80x9d and xe2x80x9cwritexe2x80x9d functions, rendering such electrodes useful in optical information storage and processing.
The prior art believed to be relevant as a background to the present invention consists of the following:
1. Degani, Y., Heller, A., J. Am. Chem. Soc., 110:2615, 1988.
2. Willner, I., Katz, E., Riklin, A., Kasher R., J. Am. Chem. Soc., 114:10965, 1992.
3. Willner et al., U.S. Pat. No. 5,443,701.
4. Lion-Dagan, M . Katz, E., Willner, I., J. Amer. Chem. Soc., 116791 3, 1994.
5. Willner, I., Lion-Dagan, M., Marx-Tibbon, S., Katz, E., J. Amer. Chem. Soc., 117:6581, 1995.
6. Willner, I. and Rubin, S., Angen. Chem. Int. Ed. Engl. 35: 367 , 1996.
7. Willner, I., Riklin, A., Shoham, B., Rivenson, D., Katz, E., Adv. Mater., 5:912, 1993.
8. Massey, V., Hemmerich, P., in Flavins and Flavoproteins, V. Massey and C. H. Williams (Eds.), Elsevier, Amsterdam, 83-96, 1982.
9. Walsh, C., Fisher, J., Spencer, R., Graham, D. W., Ashton, W. T., Brown, J. E., Brown, R. D., Rogers, E. F., Biochemistry, 78:1942, 1978.
10. Bxc3xcckmann, A. F., Erdmann, H., Pietzch, M., Hall, J. M., Bannister, J. V. in K. Kuneoyagi (Ed.), Flavins and Flavoproteins, Gruyter, Berlin, p. 597, 1994.
11. Riklin, A., Katz, E., Willner, I., Stocker, A., Bxc3xcckmann, A.F., Nature, 376:672, 1995.
12. Willner, I., Liondagan, M., Marxtibbon S., Katz, E., J. Amer. Chem. Soc., 117: 6581, 1995
13. Namba, K., Suzuki, S., Bull. Chem. Soc. Jpn., 48:1323, 1975.
14. Katz, E., Schlereth, D. D., Schmidt, H. L., J. Electroanal. Chem., 367:59, 1994.
Covalent coupling of redox active groups (ferrocene, bipyridinium, etc.) to amino acid residues of redox enzymes produces biocatalysts that electrically communicate with electrodes electrically xe2x80x9cwiredxe2x80x9d enzymes(1-3). Enzymes modified by photoisomerizable groups (e.g. nitrospiropyran/nitromerocyanine) show different enzymatic activities for the different light-induced generated photoisomer states(4,5). The use of photoswitchable biocatalysts as active matrices for optical recordings and optobioelectric devices was recently reviewed(6).
Electrically-wired enzymes were employed for the determination of analytes in electrochemical cells by the attachment of the electrobiocatalyse to electrodes(3,7). In all of the described systems, the functional electroactive or photoactive units are randomly distributed around the protein. The effectiveness of electrical contact between the enzyme redox-center and the electrode is limited. As a result, the rate of electron transfer between the enzyme redox center is relatively slow. This results in competitive electron transfer reactions with co-substrates (e.g. oxygen) or interfering substrates (e.g. oxidation of uric acid or ascorbic acid). As a result the magnitude of the resulting currents that assay the respective analytes are moderately low and the analysis had to be performed in an oxygen free environment. Special care had to be made to eliminate any interfering reagents from the analysis medium.
For many enzymes (e.g. flavoenzymes) the FAD-cofactor can be removed from the native protein to yield the unfolded apo-protein which can be reconstituted back with the natural cofactor or chemically modified FAD cofactors to yield the bioactive enzyme(8-10). The reconstitution of apo-flavoenzymes with a FAD-cofactor bound to an electron mediator group generated an xe2x80x9celectro enzynzexe2x80x9d that exhibited electrical contact with electrode surfaces. Mediated electron transfer activates the reconstituted enzymes for the electrocatalytic oxidation of their substrates(11).
Enzyme-electrodes for electrochemical determination of an analyte can operate as non-invasive or invasive analytical devices. For invasive analyses the electrodes must be constructed of bio compatible non-hazardous substances, and the electrodes must be fabricated as thin needles to exclude pain upon invasive penetration. The low surface area of the electrodes must be compensated by a high electrical activity of the sensing biocatalysts to yield measurable current responses.
The functions of enzymes modified by randomly substituted photoisomerizable units are only incompletely switched by external light signals. The perturbation structure of the protein environment of the active redox center of enzymes is only partially affected by remote photoisomerizable units. This yields only to partial, incomplete, deactivation of the photoisomerizable enzyme(12).
It is an object of the present invention to provide an electrochemical method and system for the determination of the presence and optionally the concentration of an analyte in a liquid medium.
It is furthermore an object of the invention to provide electrodes for use in such method and system. It is particularly an object of the invention to provide such electrodes comprising a solid, electrically conducting matrix carrying immobilized enzymes such that electric charge and flow between the electrode to the enzymes renders the enzyme catalytically active whereby they catalyze a reaction in which the analyte to be assayed is converted into a product.
It is furthermore an object of the invention to provide such electrodes with high and efficient electron transport between the electrode and the enzymes such that the electrode is essentially insensitive to the presence of otherwise interfering redox reagents, i.e. there is a minimum of non-specific redox reactions.
It is furthermore an object of the invention to provide enzyme-electrodes where the entities immobilized on the electrodes are non toxic and non immunogenic enabling the use of the electrode in invasive analysis.
It is furthermore an object of the invention to provide enzyme-electrodes with photoswitchable enzymes immobilized on the electrode surface, for use in the recordal of optical signals and transduction of recorded optical signals.
It is another object of the invention to provide uses of the electrodes of the invention as well as processes for their preparations.
Other objects of the invention will be clarified from the description below.
The present invention has two aspects: one aspect, to be referred to herein as the xe2x80x9cfirst aspectxe2x80x9d in which the electrode is useful for the determination of the presence and optionally the concentration of an analyte in a liquid medium; and another aspect, to be referred to herein as the xe2x80x9csecond aspectxe2x80x9d, in which the electrical response of the electrode is photoregulated (i.e. the degree of electrical response is controllable by irradiation of light at a specific wavelength) allowing the use of the electrode in recordal of optical signals and the electrical transduction of recorded optical signals. Both aspects of the present invention share a common denominator in that the electrodes carry immobilized enzymes, and in that the enzymes have functionalized cofactors, i.e., cofactors modified by the addition of a functional group or moiety (such enzymes to be referred to at times as xe2x80x9cfunctionalized enzymzesxe2x80x9d).
In accordance with the invention a functionalized enzyme may be obtained by reconstituting an apo-enzyme (an enzyme without its cofactor) with a FAD modified by the addition of a functional group or moiety (xe2x80x9cfunctionalized FADxe2x80x9d).
In accordance with one embodiment the functional group or moiety is a binding moiety capable of chemical association with, attachment to or being chemically sorbed onto the surface of the electrode. In accordance with another embodiment, the functional group or moiety is an electron mediator group which is a group capable of reversibly changing its redox state and transfer electrons to and from the FAD. In accordance with a further embodiment the functionalized group is a photoisomerizable group which can change its isomerization state upon photostimulation. It is possible also in accordance with other embodiments of the invention for the functionalized FAD to have more than one functional group or moiety, e.g. a binding moiety and an electron mediator group, or a binding moiety and a photoisomerizable group, etc.
In the case of a functionalized FAD having an electron mediator group, and particularly such wherein the functionalized FAD has both a binding moiety and an electron mediator group, there is a highly efficient electron transfer between the electrode and the FAD, yielding enzyme turnover rate which approaches maximal theoretical considerations. Such an electrode which is useful particularly in accordance with the first aspect of the invention, gives rise to a very high electrical response to a change in analyte concentration. Furthermore, the high turnover rate renders the electrode essentially insensitive to interfering agents such as non-specifically oxidizing or reducing agents, e.g. oxygen, ascorbic acid, uric acid, etc.
In accordance with the first aspect of the invention, the functionalized FAD preferably comprises an electron mediator group. It should be noted that where the functionalized FAD in the functionalized enzyme used in the first aspect does not comprise an electron mediator group, there is an electron mediator group which may be freely tumbling in solution or independently immobilized on the surface of the electrode, side by side with the modified FAD.
In accordance with the second aspect of the invention, wherein the functionalized FAD has a photoisomerizable group, enzymes have two catalytic states representing xe2x80x9cONxe2x80x9d and xe2x80x9cOFFxe2x80x9d states. This allows the xe2x80x9cwritingxe2x80x9d of a photo event on the surface of the electrode which is then xe2x80x9cmemorizedxe2x80x9d by the electrode by means of the induced photoisomerizable state of the functionalized cofactor, and this state can then be xe2x80x9creadxe2x80x9d by the electrode by measuring a change in the electrical response.
In the method and system of the invention, the changes in the analyte""s concentration in the case of the first aspect or a change in the photoisomerization state in the case of the second aspect gives rise to a change in the electrical response. The term xe2x80x9celectrical responsexe2x80x9d which is used herein denotes the current-voltage behavior of an electrode, e.g. the current response or the flow of charge of an electrode under a certain applied potential, etc. The electrical response may be determined by measuring current or charge flow, under alternating current or direct current conditions.
In the following the term xe2x80x9cdeterminexe2x80x9d or xe2x80x9cdeterminationxe2x80x9d will be used to denote both determination of only the presence or determination of both the presence and concentration of an analyte in a liquid medium.
In the following, use will also be made of the term xe2x80x9creconstitutionxe2x80x9d referring to the joining together of an apo-enzyme (enzyme without its cofactor) with a cofactor to obtain a functionalized enzyme. In accordance with the invention the reconstitution of the enzyme is performed with a synthetic, functionalized FAD-cofactor. The term xe2x80x9creconstituted functionalized enzymexe2x80x9d will be used to denote an enzyme obtained by reconstitution of an apo-enzyme with a functionalized cofactor.
The functionalized enzyme reconstituted with a functionalized FAD will have properties which will be influenced by the type of the FAD modification. For example, a functionalized enzyme having a FAD comprising a linking group with a binding moiety and having an electron mediator group will be electrobiocatalytically active and capable of directly receiving electrons from or transferring electrons to the electrode (depending on whether it catalyzes in a reduction or oxidation pathway, respectively), without the need for a separate electron mediator group. Such a functionalized enzyme exhibits a highly efficient electrical contact with the electrode with an enzyme turnover approaching maximal theoretical consideration. A functionalized enzyme with a functionalized FAD having a photoisomerizable group will have different electrobiocatalytic properties, depending on the isomerization state of the photoisomerizable group; the catalytic properties of the enzyme can thus be controlled by light.
In accordance with the teaching of the invention there is thus provided an electrode carrying FAD-dependent enzymes on its surface, the enzymes having a functionalized FAD, being an FAD modified by the addition of a functional group or moiety, being one or more of the group consisting of:
(a) a binding moiety which can chemically associate with, attach to or chemically sorb onto the electrode, the enzyme being immobilized on the electrode by binding of the binding group to the electrode""s surface;
(b) an electron mediator group which can transfer electrons between the surface of the electrode and the FAD; and
(c) a photoisomerizable group which can change from one isomerization state to another by exposure to light of a first wavelength, such photoisomerization either increases or decreases the electrically induced catalytic activity.
Preferred electrodes for use in accordance with the first aspect of the invention are such wherein the functionalized FAD comprises both a binding moiety and an electron mediator group. Typically the electron mediator group will be sandwiched between the binding moiety and the remainder of the functionalized FAD thus allowing efficient and rapid electron transfer between the surface of the electrode and the FAD.
In electrodes for use in accordance with the second aspect of the invention the functionalized enzymes may at times be bound to the electrode by means of a group linked to a surface residue of the protein at its one end and having a binding moiety at its other end. Alternatively, the functionalized FAD may comprise both a photoisomerizable group and a binding moiety bound to the electrode.
The present invention also provides a process for preparing an electrode having FAD-dependent redox enzymes immobilized thereon, the process comprising:
(a) preparing apo-enzymes by treating an FAD-dependent enzyme so as to remove the FAD-cofactor therefrom;
(b) preparing a functionalized FAD by covalent binding to a binding moiety capable of chemical association with, attachment to or a chemical sorption to the surface of the electrode;
(c) reacting the functionalized FAD with the electrode under conditions such that the modified FAD becomes immobilized onto the electrode through chemical association, attachment or sorption of the binding moiety onto the surface of the electrode; and
(d) reacting the electrode obtained in (c) with the apo-enzyme under conditions in which the apo-enzyme combines with the modified FAD to yield functional immobilized enzymes.
As will be appreciated, in the above process, steps (a) and (b) can be reversed. Furthermore, it is at times possible to first combine the apo-enzyme with the modified FAD and only then immobilizing the entire complex onto the surface of the electrode.
Where the functionalized FAD comprises other functional groups, i.e. an electron mediator group or a photoisomerizable group, these may be, a priori, included in the modified FAD prior to its immobilization onto the electrode, or may be added to the modified FAD after immobilization. A preferred immobilization scheme for preparing an electrode for use in accordance with the first aspect, comprises:
(a) treating an electrode to obtain a monolayer comprising an electron mediator group, the electron mediator group having a binding moiety which is capable of chemical association with, attachment to or chemical sorption to the surface of the electrode, the treatment comprising binding of the binding moiety onto the surface of the electrode;
(b) reacting the electrode obtained in (a) with an FAD such that the FAD becomes immobilized onto the electrode through chemical attachment to the electron mediator group;
(c) reacting the electrode obtained in (b) with apo-enzyme under conditions in which the apo-enzyme combines with the FAD component of the modified FAD.
The present invention further provides, by another of its facets, an electrochemical system for determining the presence of an analyte liquid medium, the system comprising:
(a) an electrode carrying on its surface FAD-dependent enzymes, the enzymes being capable of catalyzing a redox reaction in which an analyte is converted into a product, the enzymes comprising a functionalized FAD having a binding moiety which is chemically associated with, attached to or chemically sorbed onto the surface of the electrode;
(b) an electron mediator group which can transfer electrodes between the surface of the electrode and the FAD, the electron mediator group either
(ba) forming part of or being covalently bound to the functionalized FAD,
(bb) being independently immobilized onto the surface of the electrode,
(bc) being covalently bound to the enzyme, or
(bd) being freely tumbling (i.e. being non immobilized) in a medium surrounding the electrode; and
(c) an electrical circuitry for charging the electrode and measuring the electrical response.
As will be appreciated, the analyte specificity of the system is determined by the type of the immobilized enzyme.
The present invention further provides a method for determining the presence of an analyte in a liquid medium, the method comprising:
(a) providing an electrochemical system as defined above;
(b) introducing a sample of said liquid medium into the electrochemical cell of the system;
(c) charging the electrode and measuring the electrical response, a change in the electrical response as compared to an electrical response under the same condition in a control medium which does not comprise the analyte, indicating the presence of the analyte in the system.
Electrodes in accordance with the first aspect of the invention exhibit high turnover rates which approaches theoretical concentrations and are thus essentially insensitive to various non-specific oxidizing or reducing agents such as oxygen, etc. This is particularly the case in electrodes of the invention where the electron mediator group forms part of or is covalently attached to the functionalized FAD. Such electrodes are thus suitable for performing measurement in a non protected environment. e.g., measurement performed in vivo. A particular example is an electrode in the form of a needle which can be inserted into a blood vein and continuously measure a desired parameter, e.g. glucose level. All the entities of the surface of the electrodes, i.e. the enzymes, may be made to be identical to such normally present within the body and accordingly there will typically be no immune response or any toxic effect, which may otherwise result from a continuous exposure to a foreign entity.
Electrodes and systems for continuous in vivo measurement of various parameters, are particularly preferred in accordance with the first aspect of the invention.
Enzymes which can be used in accordance with the invention include glucose oxidase (GOD), in which case the analyte will be glucose; D-aminoacid oxidase (DAAO), in which case the analyte is a D-aminoacid (e.g. D-alanine); lactate oxidase (LacOx), in which case the analyte is lactic acid; glutathione reductase (GR), in which case the analyte is oxidized glutathione; and many other flavoenzymes.
In accordance with a second aspect of the invention there is provided an electro chemical ""system for the recordal of optical signals having a first wavelength and the electrical transduction of the recorded signals, the system having an electrochemical cell comprising:
(a) an electrode carrying immobilized FAD-dependent redox enzymes, the enzyme:
(aa) having a functionalized FAD comprising a photoisomerizable group which changes its isomerization state from a first to a second state upon photostimulation of light of the first wavelength, a change in the isomerization state giving rise to a change in the rate of catalytic activity of the redox enzyme,
(ab) being immobilized onto the surface of the electrode through a linking group which either
(aba) forms part of or being covalently bound to the functionalized FAD, or
(abb) is covalently bound to an external moiety on the surface of the enzyme;
(b) an electron mediator group which can transfer electrons between the electrode and the FAD, the electron mediator group being either
(ba) freely tumbling in the medium surrounding the electrode,
(bb) independently immobilized onto the surface of the electrode,
(bc) covalently bound to the enzyme, or
(bd) covalently bound to or forming part of the modified FAD;
(c) a substrate for the catalytic activity of the enzyme; and
(d) an electric circuitry for charging the electrode and measuring the electrical response.
Preferably, the photoisomerizable group can be isomerized reversibly by exposure to light to different wavelength regions. Thus, light irradiation at a first wavelength will change the isomerization state from a first state to a second state whereas light of a second wavelength will change the isomerization state between the second state to the first state. Accordingly, the system may comprise a light source irradiating light at the second wavelength for changing the isomerization state from the second back to the first state. Thus, the system will record light events at a first wavelength and can then be reset by the second wavelength emitted from the system""s light source.
The present invention further provides, in accordance with the second aspect, a method for recordal of optical signals having a first wavelength and electrical transduction of the recorded optical signals, the method comprising:
(a) providing an electro chemical system as defined above;
(b) exposing the electrode to a light source;
(c) charging the electrode and measuring the electrical response, changing the electrical response indicating exposure to light having said first wavelength.
The present invention still further provides a process for preparing electrodes for use in accordance with the second aspect of the invention, the process comprising:
(a) preparing apo-enzyme by treating a FAD-dependent enzyme so as to remove the FAD therefrom;
(b) preparing a modified FAD by covalent binding of a group capable of attachment or binding to a photoisomerizable group;
(c) reacting the modified FAD with the photoisomerizable group to yield a photoisomerizable FAD;
(d) combining the apo-enzyme with the photoisomerizable FAD to yield a reconstituted photoisomerizable redox enzyme; and
(e) providing an electrode carrying linking groups immobilized thereon and reacting the reconstituted enzymes with the electrodes such that the enzymes become covalently bound to the linking group.
Another process to prepare an electrode in accordance with a second aspect, comprises:
(a) preparing apo-enzyme by treating a FAD-dependent enzyme so as to remove the FAD therefrom;
(b) preparing a modified FAD by covalent binding of a group capable of attachment or binding to a photoisomerizable group;
(c) reacting the electrode with a linking group having a binding moiety capable of association, chemical binding or sorption to the electrode and having a functional unit capable of binding to a photoisomerizable group, the reaction being under condition so that said binding moiety associates, chemically binds or sorbs with the surface of the electrode;
(d) reacting the electrode obtained in (c) with a photoisomerizable group;
(e) reacting the electrode obtained in (d) with the modified FAD obtained in (b), such as to obtain a monolayer comprising immobilized photoisomerizable FAD moieties on the electrode; and
(f) reacting the apo-enzymes with the electrode obtained in (e) under condition whereby the enzyme is reconstituted on the surface of the electrode thus yielding photo active redox enzymes immobilized on the electrode.
Enzymes which can be used in accordance with the second aspect of the invention include those mentioned above in connection with the first aspect.
A linking group which can be utilized in accordance with the present invention to immobilize an FAD onto the surface of an electrode, may have the following general formula (I):
Zxe2x80x94R1xe2x80x94Qxe2x80x83xe2x80x83(I) 
wherein:
Z is a binding moiety in case where the electrode is made of gold, platinum or silver, represents a sulphur-containing moiety which is capable of chemical association with, attachment to or chemisorption onto said metal; and in case where the electrode is made of glass, represent methoxy or alkoxy silane residues which are capable of chemical association, attachment to or chemisorption onto said glass;
R1 represents a connecting group;
Q is a functional group which is capable of forming a covalent bond with a moiety in the catalytic peptide or in the porphyrin group.
Z, where the electrode material is a metal, may for example be a sulphur atom obtained from a thiol group, a disulfide group, a sulphonate group, or a sulphate group.
R1 may be a covalent bond or may be a peptide or polypeptide or may be selected from a very wide variety of suitable groups such as alkylene, alkenylene, alkynylene phenyl containing chains, and many others.
Particular examples of R1 are a chemical bond or a group having the following formulae (IIa), (IIb), (IIc) or (IId) 
wherein
R2 or R3 may be the same or different and represent straight or branch alkylene, alkenylene, alkynylene having 1-16 carbon atoms or represent a covalent bond,
A and B may be the same or different and represent 0 or S,
Ph is a phenyl group which is optionally substituted, e.g. by one or more members selected from the group consisting of SO3xe2x88x92 or alkyl groups.
Q may for example be an amine group, capable of binding to a carboxyl residue; a carboxyl group, capable of binding to an amine residue; an isocyanate or isothiocyanate group or an acyl group capable of binding to an amine residue; or a halide group capable of binding to hydroxy residues of the polypeptide. Particular examples are the groups xe2x80x94NH2xe2x80x94COOH; xe2x80x94Nxe2x95x90Cxe2x95x90S; Nxe2x95x90Cxe2x95x90O; or an acyl group having the formula xe2x80x94Raxe2x80x94COxe2x80x94G wherein G is hydrogen, a halogen such as Cl, or is OH, ORb, a 
group or a 
group; Ra and Rb being, independently a C1-C12 alkyl or alkenyl or a phenyl containing chain which is optionally substituted, e.g. by halogen.
Particular examples of such a linking group are those of the following formulae (III)-(IX): 
wherein n is an integer between 1-6.
Linking the FAD with an electro mediator group or a photoisomerizable group, as well as linking of electron mediators directly onto the enzyme, may be achieved by means of a connecting group having the following formula (X):
Zxe2x80x94Fxe2x80x94R1xe2x80x94Qxe2x80x83xe2x80x83(X) 
wherein R1 has the same meaning as indicated above and Q1 and Q2 have independently one of the meanings given above for Q.
Examples of electron-mediator groups which can be used in accordance with the invention are errocene, pyrroloquinoline quinone, quinone, N,Nxe2x80x2-dialkyl-4,4xe2x80x2-bipyridinium salts and many others.
The linking group may at times comprise also another functional group, such as an electron mediator group or a photoisomerizable group. A linking group in accordance with such embodiments may have the following general formula (XI)
Q1xe2x80x94R1xe2x80x94Q2xe2x80x83xe2x80x83(XI) 
wherein Z, R1 and Q have the meaning given above, and F is the functional group.
Examples of photoisomerizable groups that can be used in accordance with the invention are nitrospiropyran, azobenzene, thiophene fulgide and many other compounds being photoisomerized from one state to the other state and back by irradiation by light of two different wavelength regions.
The present invention will now be further illustrated in the following specific embodiments and drawings.